The present invention relates generally to methods for implementing microscopy and microscopic measurements as well as a microscope and apparatus for implementing them. More particularly, the present invention relates to a method and microscope for observing phase objects such as living cells and minute pits and projections on semiconductor substrates, metal surfaces and glass substrates and a method and apparatus for measuring the number, morphology, distribution and so on of cells cultured in a culture vessel.
Living cells, being colorless and transparent, cannot be viewed through ordinary bright-field observations and, hence, are viewed by means of phase contrast microscopes or differential interference microscopes.
Like living cells, minute pits and projections occurring on semiconductor substrates or metal surfaces, too, are observed under differential interference microscopes, because of being incapable of bright-field observations.
As set forth typically in patent publication 1, a phase contrast microscope has a zonal aperture located at the pupil position of a condenser lens therein and a phase film that is similar in shape to the zonal aperture and is located at the pupil position of an objective lens conjugate to the zonal aperture via an object under observation, so that an image contrast proportional to the phase distribution of the phase object is obtainable.
As set forth typically in patent publication 2, a differential interference microscope has two doubly refracting prisms one located at the pupil position of a condenser lens and another at the pupil position of an objective lens with interference of orthogonal two polarized light components slightly displaced sideways, so that an image contrast proportional to the differentiated value of the phase distribution of a phase object is obtainable.
By the way, studies of growing and proliferating cells sampled from within a living body and applying grown cells to medical fields and pharmacometrics are now in the making.
Grown cells must be provided in a specific number and amount. So far, growing cells in a culture vessel have been observed under a microscope to visually measure how many cells are there and to what degree cells are growing. In another effort, images under observation are captured from a microscope in a CCD camera or the like and the images are processed to determine the number of cells (non-patent publication 3).
Patent Publication 1                JP(A) 7-225341        
Patent Publication 2                JP(A) 8-122648        
Patent Publication 3                JP(A) 2002-214228        
Non-Patent Publication 1                “Optics”, Vol. 30, No. 9 (2001), pp. 605 (33)-612 (40)        
Non-Patent Publication 2                “Optics” by Hiroshi Kubota, pp. 300-304 (1964, Iwanami Shoten, Publishers)        
In both the phase contrast microscope and the differential interference microscope, at the pupil position of each of an illumination optical system and an image-formation optical system there is located a device for the visualization of phase distributions. To this end, the illumination optical system must stay conjugate to the image-formation optical system. However, as the magnification of a viewing optical system goes down to make a viewing range wider, pupil aberration between the illumination optical system and the image-formation optical system becomes too large to keep the pupils in conjugate relations. This renders it difficult to use both the phase contrast microscope and the differential interference microscope for observations at magnifications lower than 4.
For instance, when living cells are observed during growing or minute pits and projections remaining on a semiconductor substrate surface, faster observations are necessary over a wide viewing range. However, both the phase contrast microscope and the differential interference microscope offer a problem in this regard.
Further, with living cells under observation by the phase contrast microscope, the coexistence of an intensity component that is not proportional to the phase difference of living cells and called a halo renders observations difficult. Furthermore, the coexistence of external perturbations such as illumination variations renders observations much harder.
With visual observations and measurements of growing cells in a culture vessel, there are problems such as longer time taken for measurements all over the culture vessel, differences from person to person, and lower repeatability.
In image processing, a cell portion is extracted depending on an image contrast difference between the cell portion to be detected and other cell-free portion. However, this is susceptible of influences by S/N changes of the detected image due to changes in the quantity distribution of illumination light or changes in viewing environments. There is another requirement that the viewing magnification be increased, ending up with another problem that the time for making measurements all over the culture vessel surface becomes long.